Cell--cell interactions are a feature of a variety of biological processes. In the activation of the immune response, for example, one of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury. The adhesion of leukocytes to vascular endothelium is an obligate step in their migration out of the vasculature (for a review, see Albelda et al., FASEE J., 1994, 8, 504). As is well known in the art, cell--cell interactions are also critical for propagation of both B-lymphocytes and T-lymphocytes resulting in enhanced humoral and cellular immune responses, respectively.
In several instances, the adhesion of one cell type to another is mediated by interactions between specific proteins, termed "adhesion molecules," located on the surface membrane of cells. The interaction between adhesion molecules is similar to classical receptor ligand interactions with the exception that the ligand is fixed to the surface of a cell instead of being soluble. One group of related (by peptide sequence), biologically significant molecules mediating cell--cell interactions are known in the art as CAMs (cellular adhesion molecules). CAMs include, for example, several intercellular adhesion molecules (i.e., ICAM-1, ICAM-2 and ICAM-3), endothelial leukocyte adhesion molecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1), and platelet endothelial cell adhesion molecule 1 (PECAM-1). The CAM family is in turn a part of the immunoglobulin superfamily of genes (Newman et al., Science, 1990, 247, 1219).
In cell:cell interactions, a given cellular adhesion molecule present on a first cell binds one or more ligands present on a second cell. For example, ICAM-1 binds LFA-1, LFA-3 binds CD2, etc. Such interactions may be simply represented as follows: EQU CELL.1--LFA-3+CD--CELL.2.fwdarw.CELL.1--LFA-3:CD2--CELL.2
The binding of a given cellular adhesion molecule to its ligand can have many results, including (1) facilitating the ability of the two cells to remain in close contact for a period of time, during which molecules (e.g., antigens) can be passed directly from one cell to the other and/or (2) initiating a cellular response in, for example, the second cell via a conformational or chemical change in the ligand of the second cell that results from the ligand's interaction with the first cell's adhesion molecule (i.e., signal transduction). In the latter instance, a free (i.e., not associated with a cell) soluble form of the appropriate adhesion molecule may be capable of binding to the surface ligands of the second cell and thereby evoking the same or similar cellular response. Such soluble isoforms of adhesion molecules may also be produced in vivo in order to competitively bind with the membrane-bound ligand and thus reduce or inhibit cell:cell interactions, and/or to effect cell:cell de-adhesion. Using LFA-3 and CD2 as examples, such reactions may be simply diagramed as follows, wherein "sCD2" indicates a soluble form of CD2 present in excess: EQU CELL.1--LFA-3:CD2--CELL.2+sCD2.fwdarw.CELL.1--LFA-3:sCD2+CD2--CELL.2
As is known in the art, cell:cell interactions play an important role in the activation of thymus-derived lymphocytes (T cells), which are regulatory and/or effector cells in a variety of immune responses. That is, some T cells (e.g., helper cells) act to regulate other cells of the immune system by, e.g., producing and releasing factors that stimulate such other cells to effect molecular immunoreactive activities. Other T cells [e.g., cytotoxic, cytolytic or natural killer (NK) cells] directly effect immunoreactive activities by, e.g., lysing target cells bearing a foreign or abnormal antigen. In either event, the stimulation and antigen specificity of T cells in an immune response is mediated by cell:cell interactions between a T cell and, e.g., an antigen presenting cell (APC) (Bierer et al., FASEB J., 1988, 2, 2584). At a molecular level, these cell:cell interactions are mediated by adhesion molecules present on T cells and APCs (Bierer et al., FASEB J., 1988, 2, 2584; Makgoba et al., Immunol. Today, 1989, 10, 417).
Several types of adhesion molecules are implicated in mediating interactions between T cells and APCs. These include at least three lymphocyte function associated antigens (LFA-1, LFA-2 and LFA-3; Sanchez-Madrid et al., Proc. Natl. Acad. Sci. U.S.A., 1982, 79, 7489) and ICAMs (including ICAM-1, ICAM-2 and ICAM-3). The present invention is drawn to modulators of LFA-3 function, the ligand/receptor of which is CD2. CD2 is expressed primarily on T cells, including helper and NK cells, while LFA-3 is expressed on all human cells except thymocytes and some T cells (Bierer et al., FASEB J., 1988, 2, 2584).
LFA-3 proteins are glycoproteins expressed on the surfaces of a variety of cell types (for reviews of LFA-3 and related proteins, see Dustin et al., Annu. Rev. Immunol., 1991, 9, 27). LFA-3 plays a role in mediating thymocyte interactions with thymic epithelial cells, and antigen-dependent and--independent interactions of T lymphocytes with target cells and APCs (Wallner et al., J. Exp. Med., 1987, 166, 923). This interaction can also enhance major histocompatibility complex (MHC) T cell recognition (Selvaraj et al., Nature, 1987, 326, 400). LFA-3 is also associated with some hyperproliferative diseases, such as myeloma (Cook et al., Acta Haematol., 1997, 97, 81; Tatsumi et al., Jpn. J. Cancer Res., 1996, 87, 837).
Furthermore, LFA-3 is upregulated in cells infected with, or enhances the replication of, certain viruses, for example, cytomegalovirus (Grundy et al., Immunol., 1993, 78, 405).
Due to LFA-3's involvement in cellular processes associated with immune responses, tumorigenesis and other disease states, it is hoped that inhibitors of LFA-3 expression would provide a novel therapeutic class of immunosuppressive and/or anti-inflammatory and/or anticancer agents with activity towards (1) autoimmune disorders such as multiple sclerosis, particularly autoimmune disorders of the thyroid such as Graves' disease, and undesired immune responses, such as, for example, those that occur in graft versus host disease (GVHD); (2) a variety of inflammatory diseases or disorders with an inflammatory or T cell-mediated component such as various forms of arthritis; allograft rejections; asthma; inflammatory diseases of the bowel, including Crohn's disease; various dermatological conditions such as psoriasis; and the like, and (3) a variety of hyperproliferative diseases or disorders including, but not limited to, cancers, tumors, and the growth and spreading (metastasis) thereof.
To date, there are no known therapeutic agents which effectively prevent the expression of LFA-3. Current agents which affect cellular adhesion molecules include monoclonal antibodies and polypeptide soluble forms of the ligands of adhesion molecules. Monoclonal antibodies to LFA-3 may prove to be useful for the treatment of acute inflammatory response due to expression of LFA-3. However, the binding of antibodies to membrane-bound LFA-3 may mimic ligand (CD2) binding and thus stimulate signal transduction, even though CD2 binding is blocked; compounds that reduce or inhibit the expression of LFA-3, such as the antisense compounds of the invention, should block both ligand and signal transduction. Moreover, with chronic treatment, the host animal develops antibodies against the monoclonal antibodies thereby limiting their usefulness. In addition, monoclonal antibodies are large proteins which may have difficulty in gaining access to the inflammatory site. Polypeptide forms of the cell adhesion molecules suffer from many of the same limitations as monoclonal antibodies in addition to the expense of their production and their low binding affinity. Moreover, LFA-3 is transmembrane or membrane-bound protein, and polypeptides derived from LFA-3 are often insoluble in aqueous solution, limiting their therapeutic potential (Dustin et al., Annu. Rev. Immuno., 1991, 9, 27). Thus, there is a long felt need for molecules which effectively inhibit LFA-3. Antisense oligonucleotides avoid many of the pitfalls of current agents used to block the effects of LFA-3. It has been found that such antisense compounds can modulate the expression of LFA-3 proteins.